Selective lysis of cells

ABSTRACT

Methods and devices for detection of micro-organisms present or suspected to be present within a mammalian blood sample are provided. A selective lysis is obtained by incubating the sample in a non-ionic detergent under alkaline conditions.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application is a divisional of U.S. National Stage Application Ser.No. 13/514,734, filed Sep. 24, 2012. Ser. No. 13/514,734 is a U.S.National Phase application under 35 U.S.C. § 371 of InternationalApplication Serial No. PCT/IB2010/055628, filed on Dec. 7, 2010, whichclaims the benefit of European Patent Application Serial No. 09178363.9,filed on Dec. 8, 2009. These applications are hereby incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to the lysis of eukaryotic cells, inparticular animal cells, such as blood cells. The present inventionfurther relates to the detection of low concentrations ofmicro-organisms such as bacteria in samples with high concentrations ofother cells.

BACKGROUND OF THE INVENTION

Molecular diagnostics aims at the rapid detection of minute amounts ofpathogens (typically bacteria) in samples such as blood. Blood ishowever a complex matrix and comprises white blood cells (leukocytes)for the adaptive immune system, red blood cells (erythrocytes) foroxygen transport, and platelets (thrombocytes) for wound healing. Thiscomplicates the direct detection of pathogens in samples such as wholeblood, which contain a high amount of cellular material.

Classical detection methods comprise the growth of bacteria on selectivemedia and/or media with indicators. Typically such assays require acultivation step of at least 1 or 2 days before identification can takeplace.

For PCR based methods the amount of bacteria in a fresh blood sample istheoretically high enough to be detected without further cultivation ofthe bacteria present within such sample. However, to allow an earlydetection of minute amounts of bacteria, large volumes of blood arerequired. The high amount of DNA in especially white blood cellsdramatically increases the background in DNA based detection methods.Also the presence of heme from hemoglobin strongly decreases theactivity of DNA polymerase. A microliter of human blood contains about4,000 to 11,000 white blood cells and about 150,000 to 400,000platelets. The concentration of DNA in blood is between 30 and 60.mu.g/ml. It is extremely challenging to detect in a volume of 10 ml ofwhole blood the presence of about 10 to 100,000 of a bacterial species.

The high amounts of DNA of the white blood cells may give rise to nonrelevant PCR products, or may scavenge the primers designed for thedetection of bacterial DNA. This necessitates a thorough DNApurification and separation of mammalian DNA before the bacterial DNAcan be detected via PCR or other methods.

Apart from interfering with the PCR reaction itself the amount ofmammalian DNA increases the viscosity of a sample. In addition, proteinsand membranes from the lysed mammalian cells form complexes whichprevent the filtration of a sample. This is particularly a problem forminiaturized devices. Further dilution of the, already large samplevolume, results in unacceptable long manipulation steps.

For the above reasons, methods to remove human DNA from a blood sampleare accordingly required.

Methods to specifically assay bacterial DNA in the presence of mammalianDNA are known. Looxter™ from the company SIRSLab uses a method to enrichmethylated DNA from a sample. As bacterial DNA is strongly methylated,this approach results in an enrichment of bacterial DNA. Molysis™ fromthe company Molzym, uses chaotropic agents and detergents to lyseselectively mammalian cells. This lysis step is followed by a digestwith a DNAse which is not affected by this chaotropic agent/detergent.Alternative approaches such as commercialized by Roche (Septifast™) relyon PCR primer pairs which are specifically designed to prevent aspecificbinding to human DNA and amplification of human DNA.

U.S. Pat. No. 6,803,208 describes a method wherein a highly dilutedsuspension of blood platelets doped with bacteria is lysed at 37.degree. C. for 15 minutes, whereafter it is possible to filter a smallamount of the lysed sample over a 0.4 .mu.m filter for visual inspectionof the bacteria which are retained on the filter. This method howeverdoes not allow to process large volumes of sample at ambienttemperatures.

SUMMARY OF THE INVENTION

Particular and preferred aspects of the invention are set out in theaccompanying independent and dependent claims. Features from thedependent claims may be combined with features of the independent claimsand with features of other dependent claims as appropriate and notmerely as explicitly set out in the claims.

One aspect of the invention relates to a method for the selective lysisof eukaryotic cells, in particular animal cells, within a samplecontaining or suspected to contain a micro-organism. This methodcomprises the steps of providing a sample with eukaryotic cells, inparticular animal cells, containing or suspected to contain amicro-organism, adding a non-ionic detergent and a buffer to the sampleto obtain a solution with a pH of about 9.5 or more, and incubating thesolution for a time period sufficiently long to lyse the eukaryoticcells, in particular animal cells, for example between 30 seconds and 10minutes, more preferably between 2 and 6 minutes. The lysis can beperformed in particular embodiments between 15 and 30 .degree. C., morepreferably around room temperature.

In particular embodiments, the sample is a mammalian blood sample, suchas whole blood.

In other particular embodiments the micro-organism is a bacterium orfungus.

According to particular embodiments, the ratio between the volume ofadded detergent and added buffer and the volume of sample is between 2/1and 1/10.

In particular embodiments, the non-ionic detergent is selected from thegroup comprising Nonidet, Brij, Tween, Igepal, reduced triton,octylglucoside, cholaat and Triton. More preferred examples are TritonX-100, Nonidet P40, Sodium deoxycholate and or Igepal Calif. 630.

In particular embodiments, the alkaline buffer as used herein has a pKavalue above 9. Examples hereof are borate, carbonate,CAPS(N-cyclohexyl-3-aminopropanesulfonic), CAPSO(3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CHES(2-(N-Cyclohexylamino)ethane Sulfonic acid), pyrophosphate andethanolamine. A particular example is sodium carbonate. The buffershould have sufficient buffer capacity that when mixed with the samplein ratios according to the present invention, the pH of the finalsolution is around 9.5 or higher.

In particular embodiments, the method further comprises the step offiltering the incubated solution on a filter with a pore size whichretains micro-organisms on the filter, such as a filter with a pore sizeof less than 0.7 .mu.m, more preferably less than 0.5 .mu.m. the methodof the present invention facilitates the filtration of high volumes ofsample without enzymatic or heat related process steps.

In particular embodiments, the method further comprises the step ofadding after the selective lysis according to the invention an acid oracidic buffer to obtain a pH between about 7 and 9, a “neutralizationstep”.

In particular embodiments, the methods as described above are followedby detection of the micro-organisms. Examples hereof are cytometry,microscopy, PCR or culturing.

In particular embodiments, the methods as described above are followedby lysis of micro-organisms.

Another aspect of the present invention relates to a device (1) for thedetection of micro-organisms in sample, comprising: a lysis chamber (2)for accepting a sample fluid with a volume below 40 ml, preferably below20 ml and more preferably between 1 and 20 ml, a reservoir (3)comprising an alkaline buffer with a pH of about 9.5 or more andcomprising a non-ionic detergent, or a reservoir comprising an alkalinebuffer (31) with a pH of about 9.5 or more, a reservoir comprising anon-ionic detergent (32), connected to the lysis chamber, a filter (4)connected to the lysis chamber for filtering the sample after lysis, thefilter having a pore size which retains bacteria on the filter, and adetection chamber (5) for assaying the presence of DNA.

Herein the alkaline buffer has typically a pKa above 9.5 so the finalsolution will have a pH of about 9.5 or higher, and the non-ionicdetergent is typically Triton X-100, Sodium deoxycholate, Nonidet P40and/or Igepal Calif. 630.

Methods as described in the present invention allow a selective lysis ofwhite and red blood cells in a sample while bacteria and fungi remainintact (either dead or alive).

Methods as described in the present invention make it possible toprocess a sample without substantially diluting such sample, andconsequently allow to process larger volumes of sample. In addition,there is no need for enzymatic degradation of DNA by e.g. DNase or theuse of heat, making this method less complex compared to methods knownin the prior art.

Methods as described in the present invention result in lysed sampleswith a low viscosity and a minimum of aggregates, which makes itpossible to filter large volumes of the lysed sample over a filter whichretains bacteria. Further processing of the bacteria on such filter canproceed with volumes between about 100-1000 .mu.l, which makes itpossible to process large sample volumes for subsequent procedures andto perform the required manipulations, such as neutralization andwashing, fully automated in an integrated cartridge.

The above and other characteristics, features and advantages of thepresent invention will become apparent from the following detaileddescription, taken in conjunction with the accompanying drawings, whichillustrate, by way of example, the principles of the invention. Thisdescription is given for the sake of example only, without limiting thescope of the invention. The reference figures quoted below refer to theattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the filtration efficiency of large volumes of blood afterselective lysis at different pH values in accordance with a particularembodiment of methods of the invention.

FIG. 2 shows the recovery of different bacteria after lysis at differentpH values in accordance with a particular embodiment of methods of theinvention.

FIG. 3 shows the recovery of different bacteria after lysis at differentincubation times in accordance with a particular embodiment of methodsof the invention.

FIG. 4 shows reduction of human background DNA by selective lysisaccording to the present invention.

FIGS. 5 and 6 show detection of different types of pathogens in 1 and 5ml full blood respectively.

FIG. 7 shows a comparison between manual and device performed methodaccording to the present invention.

FIG. 8 shows comparison of the method according to the invention tocommercially available sepsis detection test

FIG. 9 shows lysis of pathogens after selective lysis and capture onfilter according to the present invention.

FIG. 10 shows pathogen lysis efficiency in comparison to other lysismethods when performed after selective lysis and capture on filteraccording to the present invention.

FIG. 11 shows a schematic overview of an embodiment of a device forperforming a selective lysis as described in embodiments of the presentinvention.

FIG. 12 shows an example of an integrated device comprising a selectivelysis unit as described in embodiments of the present invention

In the different figures, the same reference signs refer to the same oranalogous elements.

The present invention will be described with respect to particularembodiments and with reference to certain drawings but the invention isnot limited thereto but only by the claims. Any reference signs in theclaims shall not be construed as limiting the scope. The drawingsdescribed are only schematic and are non-limiting. In the drawings, thesize of some of the elements may be exaggerated and not drawn on scalefor illustrative purposes. Where the term “comprising” is used in thepresent description and claims, it does not exclude other elements orsteps. Where an indefinite or definite article is used when referring toa singular noun e.g. “a” or “an”, “the”, this includes a plural of thatnoun unless something else is specifically stated.

Furthermore, the terms first, second, third and the like in thedescription and in the claims, are used for distinguishing betweensimilar elements and not necessarily for describing a sequential orchronological order. It is to be understood that the terms so used areinterchangeable under appropriate circumstances and that the embodimentsof the invention described herein are capable of operation in othersequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in theunderstanding of the invention. These definitions should not beconstrued to have a scope less than understood by a person of ordinaryskill in the art.

DETAILED DESCRIPTION OF THE EMBODIMENTS

“Blood cells” in the context of the present invention relates tomammalian cells present in blood and includes red blood cells(erythrocytes), white blood cells (leukocytes) and blood platelets(thrombocytes).

“Whole blood” in the context of the present invention relates tounprocessed blood comprising blood plasma and cells, potentially treatedwith an anti-coagulant.

“Sample” relates to an aqueous suspension comprising cellular materialand comprises body fluids such as lymph, cerebrospinal fluid, blood(whole blood and plasma), saliva, but also comprises e.g. the aqueousfraction of homogenized suspensions such as e.g. muscles, brain, liver,or other tissues.

“Eukaryotic” in the present invention relates to any type of eukaryoticorganism excluding fungi, such as animals, in particular animalscontaining blood, and comprises invertebrate animals such as crustaceansand vertebrates. Vertebrates comprise both cold-blooded (fish, reptiles,amphibians) and warm blooded animal (birds and mammals). Mammalscomprise in particular primates and more particularly humans.

“Selective lysis” as used in the present invention is obtained when in asample (such as blood) the percentage of micro-organism cells (such asbacterial cells) in that sample that remain intact is significantlyhigher (e.g. 2, 5, 10, 20, 50, 100, 250, 500, or 1000 time more)compared to the percentage of the eukaryotic cells from the organismfrom which the sample is collected that remain intact.

“Micro-organism” as used in the present invention relates to bacteria(gram positive and gram negative bacteria, as well as bacterial spores)and unicellular fungi such as yeast and molds, which are present in theorganism from which a sample has been collected, typically as apathogen.

A first aspect of the present invention relates to a method for theselective lysis of eukaryotic cells, in particular animal cells, withina sample, which contains or is suspected to contain micro-organisms suchas bacteria. The aim of the method is to increase the sensitivity of atest for the detection of minute amounts of bacteria in a sample (i.e.less than 10000, 1000, 100 or even less micro-organisms per ml ofsample). As explained in the background of the invention, DNA fromeukaryotic cells, in particular from animal cells, in a sampleinterferes with PCR based detection methods and this DNA, together withproteins and membranes form aggregates which increases viscosity afterlysis and which has a dramatic impact on the filtration of a lysedsample. To solve this problem, the eukaryotic cells, in particularanimal cells, are selectively lysed whereby a substantial part (i.e.more than 20%, 40%, 60%, 80%, 90% or even more that 95%) of themicro-organisms remains alive, or if killed by the treatment, stillcomprise the bacterial DNA within the cell wall. In methods as describedin the present invention the above mentioned problems are addressed.

Methods as described in the present invention are particularlyapplicable to any type of sample wherein the detection of DNA frommicro-organisms, particularly from bacteria, is impaired by the presenceof other cells comprising DNA, in particular cells from a host whereinthe micro-organism is present as a pathogen.

Methods as described in the present invention are now furtherillustrated for embodiments wherein the presence of minute amounts ofbacteria in a mammalian blood sample is investigated.

The blood sample can be stored as whole blood or a processed fractionsuch as plasma or a platelet preparation. Typically, methods asdescribed in the present invention are performed on freshly isolatedwhole blood. Such samples are generally treated with e.g. heparin, EDTAor citrate to avoid coagulation.

Alternatively the method is performed on fresh blood by collecting theblood from the vein directly in a tube with detergent and buffer.

Accordingly, a fresh blood sample or a preserved sample is supplementedwith a buffer and a non-ionic detergent. The selection of the buffer andits concentration are chosen in order to compensate the bufferingcapacity of the blood sample provided and to obtain a pH around orhigher than 9.5, more particular between 9.5 and 11.5, even moreparticular between 9.5 and 10.5. pH values above 11.5 are suitable formore robust organisms such as gram positive bacteria and fungi. Equallythe buffer is sufficiently concentrated such that at most a buffervolume of 200%, 150%, 100%, 50%, 20% or 10% of the sample volume isadded to the sample to obtain the required change in pH.

Suitable buffers in the context of the present invention typically havea pKa above 9, above 9.5 or even above 10 and include borate, carbonate,CAPS, CAPSO, CHES, pyrophosphate, ethanolamine, and other commonly usedbuffers with an optimal buffering capacity in the above mentioned pHranges

Suitable detergents are non-ionic detergents, which at the one hand havea lytic effect on the eukaryotic cells, in particular animal cells, onlyand on the other hand have a solubilising effect on DNA and proteins.

Examples of non-ionic detergents are alkylglycosides, Brij 35 (C12E23Polyoxyethyleneglycol dodecyl ether) (15,7), Brij 58 (C16E20Polyoxyethyleneglycol dodecyl ether) (16), Genapol (13 to 19), glucanidssuch as MEGA-8, -9, -10, octylglucoside (12,6), Pluronic F127, TritonX-100 (C.sub.14H.sub.22O(C.sub.2H.sub.40).sub.n) (13,4), Triton X-114(C.sub.24H.sub.420.sub.6) (12,4), Tween 20 (Polysorbate 20) (16, 7) andTween 80 (Polysorbate 80) (15) Nonidet P40 sodium deoxycholate, reducedTriton X-100 and or Igepal Calif. 630. A particular preferred example ofa non-ionic detergent is Triton-X 100.

The most effective concentration of detergent depends from detergent todetergent, but typically is within the range of between 0.1 and 5%, moreparticularly between 0.1 and 1%. Depending from the detergent (solid orliquid) % refers to respectively w/v % or v/v %.

The incubation of a blood sample in the presence of buffer and detergentis performed within 10 minutes, preferably between 30 seconds and 10minutes and more preferably between about 1 to 3, 1-5, 1-8, 2-6 or 1-10minutes, at temperatures between 10 and 30 .degree. C., more preferablyaround room temperature.

Methods according to the present invention have the advantage that aselective lysis is obtained below 10 minutes, at temperatures below 30.degree. C. Accordingly, the methods can be generally performed atambient temperatures without the need to heat the sample.

Optionally, after the lysis the pH of the lysed sample is brought to aneutral value (i.e. between 7 and 9) by the addition of an acid oracidic buffer in a neutralization step. It was found that a lysed sampleat neutral pH could be stored for a prolonged time (up to 1, 2, 6, 12 oreven 24 hours) without further lysis of bacterial cells and withoutdramatic changes in the fluidic properties of the lysed sample.

Another parameter investigated in the methods of the present inventionis the evaluation of the fluidic properties of the blood sample afterlysis. This can be determined by verifying which volume of lysed bloodcan be filtered through a 0.22 .mu.m filter. Methods in accordance withthe present invention allow the filtration of at least 2, 5, 7, 5 oreven 10 ml of whole blood which was diluted by addition of 1 volumes ofbuffer/detergent solution to 1 volume of sample.

Generally, methods in accordance with the present invention comprise astep wherein the intact bacterial cells are separated from the sample,typically performed by centrifugation or filtration. In particularembodiments intact bacteria are separated from the sample by passage ofthe lysed sample over a filter, with a pore size below 1 .mu.m, toretain bacteria which have typically a size between 0.5 and 10 .mu.m,such as commercially available filters with a pore size of 0.4 or 0.22.mu.m. For the filtration of samples, a wide variety of commerciallyavailable devices exists, such as filters adapted to fit on a syringesuch that after lysis within in syringe, the fluid can be passed overthe filter by manual pressure on the plunger of the syringe.

Hereafter the presence of bacteria (or fungi) on the filter can beinvestigated. In particular embodiments the presence of micro-organismsis investigated by PCR. For this purpose, bacteria (or fungi) can bewashed away from the filter and further treated for PCR amplification.Alternatively the filter is rinsed with a lysis buffer to release theDNA from the micro-organisms, which is further used in a PCR reaction.

Other detection steps that can be performed by cytometry, microscopy,PCR or culturing.

The lysis of the sample, filtration and detection of micro-organisms canbe performed within one device (schematically depicted in FIG. 11).Accordingly, one aspect of the present invention relates to a device(1), comprising a lysis chamber (2) for accepting a sample fluid with avolume between 1 and 10 ml, a reservoir (3) comprising an alkalinebuffer with surfactants as described above, or a reservoir comprising analkaline buffer (31) as described above and a reservoir comprisingsurfactants (32) as described above, the reservoirs connected to thelysis chamber (2). Within the device, the lysis chamber is connected toa filter (4) for filtering the sample after lysis wherebymicro-organisms are retained on the filter. The device further compriseschannels to remove the micro-organisms from the filter and lyse them ina separate chamber. Alternatively, the device further comprises meansfor lysing micro-organisms on the filter, and channels to transfer DNAfrom lysed bacterial or fungal cells from the filter to a separatechamber. The device can further contain a DNA purification and detectionchamber (5) for assaying the presence of DNA. Typically the detectionchamber is a PCR module.

An example of a device wherein selective lysis and subsequent DNApurification and identification takes place is depicted in FIG. 12.

Other arrangements of the systems and methods embodying the inventionwill be obvious for those skilled in the art.

It is to be understood that although preferred embodiments, specificconstructions and configurations, as well as materials, have beendiscussed herein for devices according to the present invention, variouschanges or modifications in form and detail may be made withoutdeparting from the scope and spirit of this invention.

Example 1

Effect of pH on Filtration

The goal of this experiment is to assess the effect of pH of the bufferon filtration efficiency. The buffer capacity was sufficient to obtain asimilar pH in the final solution as confirmed by measuring the pH of thefinal solution using conventional techniques known to the person skilledin the art.

The buffers contained: [0071] 1M NaBorate, pH 9.0+1% Triton X-100 [0072]1M NaBorate, pH 9.5+1% Triton X-100 [0073] 1M NaCarbonate, pH 10.0+1%Triton X-100 [0074] 1M NaCarbonate, pH 10.3+1% Triton X-100 [0075] 1MNaCarbonate, pH 10.8+1% Triton X-100

1 ml of buffer was mixed with 1 ml full blood and incubated for 3minutes. Hereafter, the neutralization buffer was added and the mixturewas filtered through a size selection filter of 25 mm in diameter andwith a pore size of 0.45 .mu.m using a vacuum filtration set-up. Thevolume of blood that was able to pass the filter before it clogged wasmeasured. Results are shown in FIG. 1. This experiment demonstrates thatthe final pH value should be around 9.5 or higher to get sufficientvolumes of blood filtered for analysis of low concentrations ofpathogens.

Example 2

The effect of pH of the buffer on the recovery of intact pathogens (E.coli) after selective lysis of the blood cells is shown.

Used buffers contained: [0079] 1M NaBorate, pH 9.0+1% Triton X-100[0080] 1M NaBorate, pH 9.5+1% Triton X-100 [0081] 1M NaCarbonate, pH10.0+1% Triton X-100 [0082] 1M NaCarbonate, pH 10.5+1% Triton X-100

Identical amounts of bacteria are spiked into 1 ml blood. This volume istreated with the above-mentioned buffers for 3 min. Hereafter the bloodis centrifuged (10 min, 4000 g) to collect the intact bacteria. Bacteriaare lysed using a standard alkaline lysis method and the DNA is purifiedusing Qiagen spin columns (QiaAmp blood mini kit). The amount of DNA isquantified using real-time PCR. The result is shown in FIG. 2.

The abovementioned figure shows the recovery of the bacteria as afunction of the pH of the selective lysis buffer. At low pH values, thewhite blood cell DNA is not degraded and is inhibiting the PCR reaction.At high pH values, the bacteria start to be lysed during the selectivelysis and they are not recovered.

Example 3

Influence of Incubation Time on Recovery of Pathogens

This example demonstrates the influence of prolonged incubation of bloodwith the selective lysis buffer according to the invention on therecovery of intact pathogens. A fixed number of P. aeruginosa bacteriawas spiked into blood. 1 ml of spiked blood was mixed with 1 mlselective lysis buffer (1M NaCarbonate pH 10.0+1% Triton X-100) andincubated for 1, 2, 3, 5, 7 or 10 minutes. Hereafter, 1 ml ofneutralization buffer was added. The pathogens were collected bycentrifugation (10 min at 4000 g) and the bacterial pellet was washed.Finally, the cells were lysed by standard alkaline lysis followed by DNApurification using the QiaAmp blood mini kit. The amount of recoveredDNA was measured by real-time PCR. Results are visualized in FIG. 3 andindicate that incubation preferably is performed between 30 seconds and10 minutes.

Example 4

Reduction of Human Background by Selective Lysis According to thePresent Invention

Reduction of the amount of eukarytotic cell DNA, more specifically whiteblood cell DNA in the current method is important since when present, itwill inhibit a following PCR reaction to detect pathogen DNA or RNA. Totest for the amount of remaining background DNA, different blood samplesare processed with the selective lysis protocol according to the presentinvention and the amount of white blood cell DNA in the PCR reaction isanalyzed using the RNaseP detection kit (Applied Bio systems). The Ctvalues of these samples are compared with those obtained from 200 .mu.lblood full blood samples, where all white blood cell DNA was present.From literature it is known that the human DNA originating from 200.mu.l full blood is the maximum amount of background DNA that can betolerated by a PCR reaction without inhibition of the pathogen DNAamplification. The result of the different PCR reactions is shown inFIG. 4.

This figure shows the difference in amount of human background betweenthe 1 ml processed blood samples according to the method of the presentinvention ((1M NaCarbonate pH 10.0+1% Triton X-100) and 200 .mu.l fullblood reference samples. Different samples are processed and the PCRresults are shown as individual data points. These results demonstratethat the amount of background DNA is much lower (=higher Ct values) inthe 1 ml samples processed according to the present invention ascompared to the 200 .mu.l full blood reference samples. This resultproves that the white blood cell DNA is efficiently and sufficientlyremoved from the sample when using the method according to the presentinvention.

Example 5

The goal of this example is to demonstrate the detection of thedifferent types of pathogens from full blood by using the methodaccording to the present invention. The different types of pathogens, agram-negative (P. aeruginosa), gram-positive (S. aureus) and fungi (C.albicans) were mixed together into 1 ml blood. The blood sample wastreated with the selective lysis buffer (1 ml of a 1M NaCarbonate pH10.0+1% TX-100 solution) for 3 min followed by neutralization of the pHand filtration using a size selection filter with sufficiently smallpores to retain all cells. The filter was washed to remove the remaininginhibitors such as hemoglobin and DNA of the white blood cells.Hereafter, the cells were lysed following a standard alkaline lysisprotocol and the DNA was purified using the Qiagen blood mini kit.

The pathogenic DNA was detected by real-time PCR; the Ct value is ameasure for the amount of DNA. For quantification a small part of thespiked blood sample was plated on blood agar plate to obtain the CFUcount. The data as present in FIG. 5 show that it is possible to detectlow numbers of pathogens from full blood. The reference sample containsthe same number of bacteria in a small volume of PBS buffer which isdirectly lysed, followed by DNA purification and quantification usingreal time PCR. The reference measurements and the actual enrichmentexperiments from blood gave similar Ct values, thus demonstrating thehigh recovery rates. The negative control (blood without bacteria) showsno PCR signal.

The assay allows larger volumes of blood to be used. The experimentalset-up is identical to the previous example but the amount of blood isincreased to 5 ml. The reference sample contains the same number ofpathogens as the 5 ml blood sample but the cells remain in a smallvolume of PBS and are directly lysed. The results are represented inFIG. 6.

This experiment demonstrates the possibility to recover low number ofpathogens from large volumes of blood. The data show that theconcentration of recovered pathogen DNA is similar to the reference.Therefore it can be concluded that the majority of the pathogens remainintact during the selective lysis and the reduction in the white bloodcell DNA is effective to prevent inhibition of the pathogen PCR.

Example 6

The selective lysis method according to the present invention may beperformed in various ways, not limited to but including a manualprocedure and a procedure wherein the method is performed by a deviceaccording to the present invention (integrated procedure). The presentexample compares such an integrated procedure and a manual procedure.The manual procedure requires manual pipetting and centrifugation stepswhile the integrated procedure uses a micro-fluidic cartridge and a sizeselection filter, capable of performing all the required operations. Thebasic biochemical protocol is similar: selective lysis of the white andred blood cells using a 1M NaCarbonate+1% Triton X-100 solution followedby a neutralization step after 3 min. In the next step the mixture iseither centrifuged (manual) or filtered (integrated) and the cells arewashed and finally the DNA is released by means of a standard alkalinelysis procedure. In a last step, the DNA is purified using the Qiagenblood mini kit and detected by real-time PCR. The results of theintegrated and manual procedure can be found in the following FIG. 7.Comparable results are achieved, demonstrating that the result isindependent of the implementation format of the assay.

Example 7

In this example, the method according to the present invention isbenchmarked against a commercially available method namely the MolYsisComplete kit (Molzym). This kit uses chaotropic agents and detergents tolyse selectively mammalian cells. This lysis step is followed by adigest with a DNAse which is not affected by this chaotropicagent/detergent.

For this experiment, 1 ml blood samples were spiked with differentconcentrations of S. aureus. 1 ml blood was processed as described inExample 5 and another 1 ml was processed with the MolYsis kit accordingto the manufacturer's instructions. The Ct values are plotted againstthe concentration of cells in FIG. 8 and show that the method accordingto the present invention is at least as efficient as the known MolYsiskit without the addition of enzymes or chaotroptic salts.

Example 8

After selective lysis of blood cells and enrichment of the pathogencells on the size selection filter, alkaline lysis was employed toachieve simultaneous lysis of different pathogens on the filter to makethe DNA available for PCR analysis.

FIG. 9 shows the result of the alkaline lysis procedure performed on anintegrated cartridge. 1 ml of blood was spiked with 10.sup.6 cells of S.aureus, P. aeruginosa and C. albicans. After selective lysis of bloodcells and enrichment of pathogens on the filter, alkaline lysis wasperformed, using 200 .mu.l of a solution containing 200 mM NaOH, 0.5%SDS which is incubated at 95 .degree. C. for 10 min to obtain completelysis of the pathogens in the filter. The eluates containing thepathogen DNA were neutralized with 20 .mu.l of a 1M citric acid solutionand purified using the QIAamp DNA/Blood Mini kit. As a control sample,10.sup.6 cells of each pathogen were lysed on the bench, neutralized andpurified as described above.

For optimization and benchmarking of the alkaline lysis procedure,Candida albicans was chosen as model system since these yeast cells arewell known for their rigid cell walls which are difficult to lyse. FIG.10 compares the alkaline lysis procedure (using 50 mM NaOH, 0.25% SDS incombination with heat treatment) with other lysis methods, namely highintensity ultrasound (HiFU) treatment and a commercial kit (BD GeneOhmlysis kit). For alkaline lysis and lysis by the commercial kit, thesamples were concentrated from 1 ml to 160 and 100 .mu.l, respectively,using centrifugation. For HiFU 2 ml of cell solution was used, withoutprior concentration. After lysis, unlysed cells and debris were removedfrom the sample by centrifugation. 1 .mu.l of crude lysate was used asinput for the PCR.

The combination of NaOH and SDS is more effective for lysis than each ofthe individual compounds. An increase of the concentration of eithercompound did not further increase the lysis efficiency. Alkaline lysiswithout a heat incubation step is significantly less efficient. Lysisefficiency can be increased by incubation for 2 min at 95 .degree. C.,however, for integration of the assay into a cartridge incubation for alonger time at 70 .degree. C. is preferred.

For alkaline lysis cells were resuspended in 100 .mu.A of a lysissolution containing 50 mM NaOH and 0.25% SDS. Subsequently the sampleswere incubated for 10 min at 70 .degree. C., cooled quickly to roomtemperature and neutralized by addition of 30 .mu.l 500 mM Tris-HCl, pH7.0 (yielding a final concentration of 150 mM Tris, i.e. 3 times theNaOH concentration).

For crude lysate PCR, unlysed cells and debris were removed from thesample by centrifugation (5 min, 14,000 g). 1 .mu.l of supernatant wasadded to a 25 .mu.l PCR reaction. Detection by PCR was based on a TaqmanPCR assay targeting the rRNA gene (Apollo). The PCR reaction wasconducted in Taqman Universal mastermix (Applied Biosystems), using 500nM forward primer and 300 nM reverse primer and FAM-BHQ1 labelled probe(all oligonucleotides custom synthesized by Biolegio BV). The PCRreaction was performed in a Biorad CFX real-time PCR system. After aninitial heating step of 10 min at 95 .degree. C. to activate thehot-start polymerase, 50 cycles of 15 sec at 95 .degree. C. and 1 min at60 .degree. C. were used for amplification. Fluorescence signals weredetected in each cycle during the 60 .degree. C. step. Data analysis wasperformed with the Biorad CFX software.

The invention claimed is:
 1. A device for detection of microorganismswithin a mammalian blood sample, the mammalian blood sample comprisingwhole blood that has not been processed, the whole blood including bloodplasma and eukaryotic blood cells, the the device comprising: a lysischamber, wherein the lysis chamber is configured to receive a samplefluid, wherein the sample fluid has a volume of less than 1 ml, whereinthe sample fluid is the mammalian blood sample; a first reservoircomprising an alkaline buffer with a pH of about 9.5 or more, whereinthe first reservoir is connected to the lysis chamber; a secondreservoir comprising a non-ionic detergent, wherein the second reservoiris connected to the lysis chamber; a filter, wherein the filter isconnected to the lysis chamber; and a detection chamber; wherein thelysis chamber is configured to mix a combined volume of the alkalinebuffer and the non-ionic detergent with the sample fluid to obtain afinal solution, wherein the final solution is configured to selectivelylyse the blood cells without lysing the microorganisms, wherein thecombined volume of the alkaline buffer and the non-ionic detergent isbetween 10% and 200% of the sample volume; wherein the filter isconfigured to filter the final solution, wherein the filter has a poresize which retains microorganisms on the filter, wherein the detectionchamber is configured to detect DNA in the microorganisms retained onthe filter without cultivation of the microorganisms; wherein the firstreservoir is configured to provide the alkaline buffer to the lysischamber in a sufficient amount to provide the final solution with a pHlevel of about 9.5 to about 11.5; wherein the second reservoir isconfigured to provide the non-ionic detergent to the lysis chamber in anamount effective to provide the final solution with a definedconcentration of the non-ionic detergent; wherein: the non-ionicdetergent is a solid detergent, and the defined concentration is between0.1% w/v and 5% w/v; or the non-ionic detergent is a liquid detergent,and the defined concentration is between 0.1% v/v and 5% v/v.
 2. Thedevice according to claim 1, wherein the alkaline buffer has a pKa above9.0.
 3. The device according to claim 1, wherein the alkaline buffer isselected from the group consisting of borate, carbonate,N-cyclohexyl-3-aminopropanesulfonate,3-(cyclo-hexylamino)-2-hydroxy-1-propanesulfonate,2-(N-cyclohexylamino)ethane sulfonate, pyrophos-phate, ethanolamine, andmixtures thereof.
 4. The device according to claim 1, wherein thenon-ionic detergent is Triton X-100.
 5. The device according to claim 1,wherein the alkaline buffer has a pKa above 9.0 and the non-ionicdetergent is Triton X-100.